Nogo receptor homologues and their use

ABSTRACT

This invention relates to gene polypeptides and polynucleotides that encode proteins of the Nogo-66 receptor (NgR) family and are therefore called NgR homologue 1 (NgRH1). The invention further relates to their use in identifying compounds that may be agonists or antagonists that are potentially useful in regeneration and protection of the nervous system, and to production of NgRH1 polypeptides, derivatives, and antibodies.

FIELD OF THE INVENTION

This invention relates to gene polypeptides and polynucleotides thatencode proteins of the Nogo receptor (NgR) family and are thereforecalled NgR homologues 1 (NgRH1). The invention further relates to theiruse in identifying compounds that may be agonists or antagonists thatare potentially useful in regeneration and protection of the nervoussystem, and to production of NgRH1 polypeptides, derivatives, andantibodies.

BACKGROUND OF THE INVENTION

Re-growth of injured neurones in the adult CNS of higher vertebrates islimited due to the presence of inhibitory molecules in myelin or due tothe formation of scar tissue. Myelin derived proteins, NogoA andMyelin-Associated Glycoprotein (MAG), have been shown in the past toinhibit neurite outgrowth (Huber and Schwab (2000) Biol. Chem. 381,407-419). While NogoA is a potent neurite outgrowth inhibitor thatrestricts the capacity of axonal regeneration in vivo after injury(Bregman et al. (1995) Nature 378, 498-501, Schnell and Schwab (1990)Nature 343, 269-72), MAG was shown to inhibit neurite outgrowth invitro, depending on the age of the neurones (Mukhopadhyay et al. (1994)Neuron 13, 757-767, DeBellard et al. (1996) Mol. Cell Neurosci. 7,89-101).

NogoA is amongst three different variants (NogoA, B and C) the longestsplice product of the Nogo gene (Chen et al. (2000) Nature 403, 434-439,GrandPre et al. (2000) Nature 403, 439-444, Prinjha et al. (2000) Nature403, 383-384) and belongs to the reticulon (RTN) protein family.Neutralising antibodies and the use of different domains of NogoA havedelineated two inhibitory domains in the molecule (Chen et al. (2000)Nature 403, 434-439, GrandPre et al. (2000) Nature 403, 439-444, Prinjhaet al. (2000) Nature 403, 383-384), one corresponding to the aminoterminus of the molecule (amino-NogoA) and the other to an extracellularloop region in the C-terminus, which has been termed Nogo-66 (GrandPreet al. (2000) Nature 403, 439-444).

Myelin-Associated Glycoprotein (MAG) is a member of the immunoglobolin(Ig) family (Lai et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4337-4341,Salzer et al. (1987) J. Cell. Biol. 104, 957-965) and can either promoteor inhibit neurite outgrowth depending on the age of the neurones(DeBellard et al. (1996) Mol. Cell Neurosci. 7, 89-101). Although MAG isalso present in Schwann cells of the PNS, it gets non-restrictive toperipheral nerves due to a downregulated after lesioning of peripheralnerves (Martini and Schachner (1988) J. Cell Biol. 106, 1735-1746,Fawcett and Keynes (1990) Annu. Rev. Neurosci. 13, 43-60, Brown et al.(1991) Neuron 6, 359-370).

A receptor, denoted the Nogo-66 receptor (NgR), now appears to play apivotal role in conveying inhibitory signals from myelin associatedproteins to neurones of the CNS. It binds MAG and the oligodendrocyteprotein OMgp with similar affinity as the originally discovered ligandNogo-66 and also mediates inhibition of axonal extensions in vitro andin vivo (Fournier et al. (2001) Nature 409, 341-346, GrandPre andStrittmatter (2002) Nature 417, 547-51, Wang et al. (2002) Nature 417,941-914, Domeniconi et al. (2002) Neuron 35, 283-290 (published onlineJune 28), Liu et al. (2002) Science June 27 (epub ahead of print). It isspecifically expressed in the brain and is regulated during development(Wang et al. (2002) J. Neurosci. 22, 5505-5515). NgR is a member of theproteoglycan/leucine-rich-repeat protein family and is attached to thecell surface by a C-terminal glyosyl-phosphatidyinositol (GPI) anchor.The NgR protein sequence contains eight leucine-rich-repeats (LRR)followed by a leucine-rich-repeat C-terminus (LRRCT). These motifs arefound in a functionally and evolutionarily diverse set of proteins,including adhesion molecules and signal-transducing receptors (Kobe andDeisenhofer (1994) TIBS 19, 415-421).

Recently, a NgR antagonist peptide, comprising the N-terminal 40 aminoacids of Nogo-66, was shown to induce regeneration in spinal cord injuryand also improved functional recovery, providing a potential therapeuticfor CNS injuries (GrandPre and Strittmatter (2002) Nature 417, 547-51).

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an isolated DNA from humanorigin comprising a nucleotide sequence as set forth in SEQ ID NO: 1 andtermed human NgRH1 cDNA.

In a further aspect, the invention relates to rat NgRH1 cDNA as setforth in SEQ ID NO: 24.

A further aspect the invention relates to rat and/or human type NgRH1polypeptides.

Such polypeptides include:

-   (a) an isolated polypeptide encoded by a polynucleotide comprising    the sequence of SEQ ID NO: 1 or SEQ ID NO: 24;-   (b) an isolated polypeptide comprising a polypeptide sequence having    at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the    polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 25;-   (c) an isolated polypeptide comprising the polypeptide sequence of    SEQ ID NO: 2 or SEQ ID NO: 25;

(d) an isolated polypeptide having at least 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO: 2 orSEQ ID NO: 25;

-   (e) the polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 25; and-   (f) an isolated polypeptide having or comprising a polypeptide    sequence that has an Identity Index of 0.80, 0.85, 0.90, 0.95, 0.96,    0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID    NO: 2 or SEQ ID NO: 25;-   (g) fragments and variants of such polypeptides in (a) to (f).

Also provided are nucleic acid sequences comprising at least about 15bases, preferably at least about 20 bases, more preferably a nucleicacid sequence comprising about 30 contiguous bases of SEQ ID NO: 1 orSEQ ID NO: 24. Also within the scope of the present invention arenucleic acids that are substantially similar to the nucleic acid withthe nucleotide sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 24.In a preferred embodiment, the isolated DNA takes the form of a vectormolecule comprising the DNA as set forth in SEQ ID NO: 1 or SEQ ID NO:24.

In a second aspect, the invention provides an isolated polypeptide withan amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 25.Fragments of the isolated polypeptide with an amino acid sequence as setforth in SEQ ID NO: 2 or SEQ ID NO: 25 will comprise polypeptidescomprising from about 5 to 410 amino acids, preferably from about 10 toabout 400 amino acids, more preferably from about 20 to about 100 aminoacids, and most preferably from about 20 to about 50 amino acids. Inaccordance with this aspect of the invention there are provided novelpolypeptides of human origin as well as biologically, diagnostically ortherapeutically useful fragments, variants and derivatives thereof,variants and derivatives of the fragments, and analogs of the foregoing.

In a third aspect the invention provides the use of modulators of NgRH1as therapeutic agents. Modulators described herein, include but are notlimited to agonists, antagonists, suppressors and inducers of NgRH1.

In a further aspect of the invention there are provided nucleotideprobes that are useful for detection of mRNA of the NgRH1 and anti-sensepolynucleotides that regulate translation of NgRH genes; in anotherembodiment, double stranded RNAs provided that can regulate thetranscription of NgRH1 genes. This includes small interfering RNAs(siRNAs) according to standard procedures (Zamore et al. (2000) Cell101, 25-33; Elbashir et al. (2001) Nature 411, 494-498).

Another aspect of the invention provides a process for producing theaforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods for producing the aforementioned human NgRH1polypeptides comprising culturing host cells having incorporated thereinan expression vector containing an exogenously-derived NgRH1-encodingpolynucleotide under conditions sufficient for expression of NgRH1polypeptides in the host and then recovering the expressed polypeptide.

In accordance with another aspect of the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for, inter alia,research, biological, clinical and therapeutic purposes.

In certain additional preferred embodiments of this aspect of theinvention there are provided an antibody or a fragment thereof whichspecifically binds to a polypeptide that comprises the amino acidsequence set forth in SEQ ID NO: 2 or SEQ ID NO: 25, i.e., human or ratNgRH1. In certain particularly preferred embodiments in this regard, theantibodies are highly selective for human NgRH1 polypeptides or portionsof human NgRH1 polypeptides.

In a further aspect, an antibody or fragment thereof. Is provided thatbinds to a fragment or portion of the amino acid sequence set forth inSEQ ID NO: 2 or SEQ ID NO: 25.

In another aspect, there are provided methods for treatment of diseases,disorders or damage which ultimately result in damage of the nervoussystem in a subject, where the disease is mediated by or associated withan increase or decrease in NgRH1 gene expression or an increase ordecrease in the presence of NgRH1 polypeptide in all major brain regions(except pons), skeletal muscle and liver. Such diseases, disorders ordamage include, but are not limited to, central nervous system (CNS)trauma (e.g. spinal cord or brain injuries), infarction, infection,malignancy, exposure to toxic agents, nutritional deficiency,paraneoplastic syndromes, and degenerative nerve diseases (including butnot limited to Alzheimer's disease, Parkinson's disease, Huntington'sChorea, multiple sclerosis, amylotrophic lateral sclerosis, andprogressive supra-nuclear palsy). The treatment may be achieved byadministering compounds that interfere with NgRH1 activity (e.g.antibodies to NgRH1, anti-sense nucleic acids of NgRH1 (siRNAs occordingto Zamore et al. (2000) Cell 101, 25-33 or Elbashir et al. (2001) Nature411, 494-498), NgRH1 ribozymes or chemical groups that bind to theactive site of NgRH1.

Another aspect is directed to pharmaceutical compositions comprising anantibody that binds to NgRH1 polypeptides or a fragment thereof for thetreatment of acute and chronic neurodegenerative diseases (e.g. asmentioned above), trauma and degenerative eye diseases, brain and spinaltrauma, stroke, spinal cord injuries.

In yet another aspect, the invention is directed to methods for theidentification of molecules that can bind to NgRH1 polypeptides and/ormodulate the activity of NgRH1 polypeptides or molecules that can bindto nucleic acid sequences that modulate the transcription or translationof NgRH1 polypeptides. Such methods are disclosed in, e.g., U.S. Pat.No. 6,043,024, incorporated by reference herein in its entirety.Molecules identified by such methods also fall within the scope of thepresent invention.

In yet another aspect, the invention provides cells which can bepropagated in vitro, preferably vertebrate cells, which are capable upongrowth in culture of producing a polypeptide that comprises the aminoacid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 25 or fragmentsthereof, where the cells contain transcriptional control DNA sequences,other than human NgRH1 transcriptional control sequences, where thetranscriptional control sequences control transcription of DNA encodinga polypeptide with the amino acid sequence according to SEQ ID NO: 2 orSEQ ID NO: 25 or fragments thereof.

In another aspect, the present invention provides a method for producingNgRH1 polypeptides which comprises culturing a host cell havingincorporated therein an expression vector containing anexogenously-derived NgRH1-encoding polynucleotide under conditionssufficient for expression of NgRH1 polypeptides in the host cell,thereby causing the production of an expressed polypeptide, andrecovering the expressed polypeptide.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited hereinare hereby incorporated by reference in their entirety.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

As used herein, “differentially expressed gene” refers to (a) a genecontaining at least one of the DNA sequences disclosed herein (e.g., asshown in SEQ ID NO: 1 or SEQ ID NO: 24); (b) any DNA sequence thatencodes the amino acid sequence encoded by the DNA sequences disclosedherein (e.g., as shown in SEQ ID NO: 2 or SEQ ID NO: 25); or (c) any DNAsequence that is substantially similar to the coding sequences disclosedherein. For example, the invention provides NgRH1 genes and theirencoded proteins of many different species. In specific embodiments, theNgRH1 genes and proteins are from vertebrates, or more particularly,mammals. In a preferred embodiment of the invention, the NgRH1 gene andproteins are from human origin. In its broadest sense, the term“substantially similar”, when used herein with respect to a nucleotidesequence, means a nucleotide sequence corresponding to a referencenucleotide sequence, wherein the corresponding sequence encodes apolypeptide having substantially the same structure and function as thepolypeptide encoded by the reference nucleotide sequence, e.g. they arecapable of displaying one or more known functional activities (e.g.preventing regeneration of neurons in the spinal cord or brain,conferring to a substrate the property of restricting growth, spreading,and migration of neural cells, and neoplastic cells, inhibiting dorsalroot ganglia neurite outgrowth, inducing dorsal root ganglia growth conecollapse, blocking NIH 3T3 cell spreading in vitro, blocking PC12neurite outgrowth, restricting plasticity) associated with a full-length(wild-type) NgRH1 protein. Desirably the substantially similarnucleotide sequence encodes the polypeptide encoded by the referencenucleotide sequence. The percentage of identity between thesubstantially similar nucleotide sequence and the reference nucleotidesequence desirably is at least 80%, more desirably at least 85%,preferably at least 90%, more preferably at least 95, 96, 97, 98%, stillmore preferably at least 99%. Sequence comparisons are carried out usinga Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M. S.Introduction to Computational Biology: Maps, sequences and genomes.Chapman & Hall. London: 1995. ISBN 0412-99391-0). A nucleotide sequence“substantially similar” to reference nucleotide sequence hybridizes tothe reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C., yet still encodes a functionally equivalent geneproduct.

Inhibition of the activity mediated by NgRH1 proteins can permitregeneration of neurons in the spinal cord or brain; confer to asubstrate the property of permissive growth; the spreading and migrationof neural cells and neoplastic cells; allow dorsal root ganglia neuriteoutgrowth; induce dorsal root ganglia growth cone growth; permit NIH 3T3cell spreading in vitro; permit PC12 neurite outgrowth and plasticity.

A “host cell,” as used herein, refers to a prokaryotic or eukaryoticcell that contains heterologous DNA that has been introduced into thecell by any means, e.g., electroporation, calcium phosphateprecipitation, microinjection, transformation, viral infection, and thelike.

“Heterologous” as used herein means “of different natural origin” orrepresent a non-natural state. For example, if a host cell istransformed with a DNA or gene derived from another organism,particularly from another species, that gene is heterologous withrespect to that host cell and also with respect to descendants of thehost cell which carry that gene. Similarly, heterologous refers to anucleotide sequence derived from and inserted into the same natural,original cell type, but which is present in a non-natural state, e.g. adifferent copy number, or under the control of different regulatoryelements.

“Identity” reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotide or two polypeptide sequences, respectively, overthe length of the sequences being compared.

“% Identity”—For sequences where there is not an exact correspondence, a“% identity” may be determined. In general, the two sequences to becompared are aligned to give a maximum correlation between thesequences. This may include inserting “gaps” in either one or bothsequences, to enhance the degree of alignment. A % identity may bedetermined over the whole length of each of the sequences being compared(so-called global alignment), that is particularly suitable forsequences of the same or very similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length.

“Similarity” is a further, more sophisticated measure of therelationship between two polypeptide sequences. In general, “similarity”means a comparison between the amino acids of two polypeptide chains, ona residue by residue basis, taking into account not only exactcorrespondences between pairs of residues, one from each of thesequences being compared (as for identity) but also, where there is notan exact correspondence, whether, on an evolutionary basis, one residueis a likely substitute for the other. This likelihood has an associated“score” from which the “% similarity” of the two sequences can then bedetermined.

Methods for comparing the identity and similarity of two or moresequences are well known in the art. Thus for instance, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available fromGenetics Computer Group, Madison, Wis., USA), for example the programsBESTFIT and GAP, may be used to determine the % identity between twopolynucleotides and the % identity and the % similarity between twopolypeptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in AppliedMathematics, 2, 482-489, 1981) and finds the best single region ofsimilarity between two sequences. BESTFIT is more suited to comparingtwo polynucleotide or two polypeptide sequences that are dissimilar inlength, the program assuming that the shorter sequence represents aportion of the longer. In comparison, GAP aligns two sequences, findinga “maximum similarity”, according to the algorithm of Neddleman andWunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to comparingsequences that are approximately the same length and an alignment isexpected over the entire length. Preferably, the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3, forpolynucleotide sequences and 12 and 4 for polypeptide sequences,respectively. Preferably, % identities and similarities are determinedwhen the two sequences being compared are optimally aligned. Otherprograms for determining identity and/or similarity between sequencesare also known in the art, for instance the BLAST family of programs(Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul S F et al,Nucleic Acids Res., 25:389-3402, 1997, available from the NationalCenter for Biotechnology Information (NCBI), Bethesda, Md., USA andaccessible through the home page of the NCBI at www.ncbi.nlm.nih.gov)and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; PearsonW R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448, 1988,available as part of the Wisconsin Sequence Analysis Package).

Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S andHenikoff J G, Proc. Nat. Acad. Sci. USA, 89, 10915-10919, 1992) is usedin polypeptide sequence comparisons including where nucleotide sequencesare first translated into amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to areference polynucleotide or a polypeptide sequence, the query and thereference sequence being optimally aligned and the parameters of theprogram set at the default value, as hereinbefore described.

A vector molecule is a nucleic acid molecule into which heterologousnucleic acid may be inserted which can then be introduced into anappropriate host cell. Vectors preferably have one or more origin ofreplication, and one or more site into which the recombinant DNA can beinserted. Vectors often have convenient means by which cells withvectors can be selected from those without, e.g., they encode drugresistance genes. Common vectors include plasmids, viral genomes, and(primarily in yeast and bacteria) “artificial chromosomes.”

“Plasmids” generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure. The term “isolated” means that the material is removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

As used herein, the term “transcriptional control sequence” refers toDNA sequences, such as initiator sequences, enhancer sequences, andpromoter sequences, which induce, repress, or otherwise control thetranscription of protein encoding nucleic acid sequences to which theyare operably linked.

The term “polypeptide” is used interchangeably herein with the terms“polypeptides” and “protein(s)”.

As used herein, a “chemical derivative” of a polypeptide of theinvention is a polypeptide of the invention that contains additionalchemical moieties not normally a part of the molecule. Such moieties mayimprove the molecule's solubility, absorption, biological half life,etc. The moieties may alternatively decrease the toxicity of themolecule, eliminate or attenuate any undesirable side effect of themolecule, etc. Moieties capable of mediating such effects are disclosed,for example, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

The invention includes nucleic acid molecules, preferably DNA molecules,such as (1) an isolated DNA comprising a nucleotide sequence as setforth in SEQ ID NO: 1 or SEQ ID NO: 24, (2) isolated DNA's that comprisenucleic acid sequences that hybridize under high stringency conditionsto the isolated DNA as set forth in SEQ ID NO: 1 or SEQ ID NO: 24, and(3) nucleic acid sequences that hybridize to (1) or (2), above. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).Suitable ranges of such stringency conditions for nucleic acids ofvarying compositions are described e.g. in Krause and Aaronson (1991)Methods in Enzymology, 200:546-556.

These nucleic acid molecules may act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target gene nucleic acidsequences.

The invention also encompasses (a) vectors that contain any of theforegoing coding sequences and/or their complements (i.e., antisense);(b) expression vectors that contain any of the foregoing codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; and (c) genetically engineeredhost cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The invention includes fragments of any of the nucleic acid sequencesdisclosed herein. Fragments of the full length NgRH1 gene may be used asa hybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to theNgRH1 gene or similar biological activity. Probes of this typepreferably have at least about 30 bases and may contain, for example,from about 30 to about 50 bases, about 50 to about 100 bases, about 100to about 200 bases, or more than 200 bases. The probe may also be usedto identify a cDNA clone corresponding to a full length transcript and agenomic clone or clones that contain the complete NgRH1 gene includingregulatory and promoter regions, exons, and introns. An example of ascreen comprises isolating the coding region of the NgRH1 gene by usingthe known DNA sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

In addition to the gene sequences described above, homologs of suchsequences, as may, for example be present in other species, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Further, theremay exist genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of suchgene products. These genes may also be identified via similartechniques.

For example, the isolated differentially expressed gene sequence may belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions will be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. Alternatively, the labeled fragment may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. Such low stringency conditions willbe well known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived.

Further, a previously unknown differentially expressed gene-typesequence may be isolated by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the gene of interest. The template for the reaction maybe cDNA obtained by reverse transcription of mRNA prepared from human ornon-human cell lines or tissue known or suspected to express adifferentially expressed gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a differentiallyexpressed gene-like nucleic acid sequence. The PCR fragment may then beused to isolate a full length cDNA clone by a variety of methods. Forexample, the amplified fragment may be labeled and used to screen abacteriophage cDNA library. Alternatively, the labeled fragment may beused to screen a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or issue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated.

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one activity of human or rat NgRH1.

A variety of host-expression vector systems may be utilized to expressthe differentially expressed gene coding sequences of the invention.Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit the differentiallyexpressed gene protein of the invention in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing differentially expressed geneprotein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing the differentiallyexpressed gene protein coding sequences; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containingthe differentially expressed gene protein coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid transformation vectors (e.g. Tiplasmid) containing differentially expressed gene protein codingsequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothioneine promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for thedifferentially expressed gene protein being expressed. For example, whena large quantity of such a protein is to be produced, for the generationof antibodies or to screen peptide libraries, for example, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited, to the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791), in which the differentially expressed gene proteincoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (e.g. Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109);and the like. PGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The PGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene protein can be released from the GST moiety.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“cat”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lad and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-I promoter.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is one of several insect systems that can be used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.The differentially expressed gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of differentiallyexpressed gene coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (E.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051). In mammalianhost cells, a number of viral-based expression systems may be utilized.In cases where an adenovirus is used as an expression vector, thedifferentially expressed gene coding sequence of interest may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing differentially expressed gene proteinin infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad.Sci. USA 81:3655-3659). Specific initiation signals may also be requiredfor efficient translation of inserted differentially expressed genecoding sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire differentially expressedgene, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the differentially expressed gene coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host per se are routine skills in the art.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed gene protein may be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the differentially expressed gene protein. Such engineeredcell lines may be particularly useful in screening and evaluation ofcompounds that affect the endogenous activity of the differentiallyexpressed gene protein.

When used as a component in assay systems such as those described below,the differentially expressed gene protein may be labeled, eitherdirectly or indirectly, to facilitate detection of a complex formedbetween the differentially expressed gene protein and a test substance.Any of a variety of suitable labeling systems may be used including butnot limited to radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable calorimetric signal or light when exposed tosubstrate; and fluorescent labels.

Where recombinant DNA technology is used to produce the differentiallyexpressed gene protein for such assay systems, it may be advantageous toengineer fusion proteins that can facilitate labeling, immobilizationand/or detection.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to either a differentially expressedgene product.

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed geneepitopes. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a fingerprint,target gene in a biological sample, or, alternatively, as a method forthe inhibition of abnormal target gene activity. Thus, such antibodiesmay be utilized for regeneration and sprouting and functional recoveryof the nervous system.

For the production of antibodies to a differentially expressed gene,various host animals may be immunized by injection with a differentiallyexpressed gene protein, or a portion thereof. Such host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection withdifferentially expressed gene product supplemented with adjuvants asalso described above. Monoclonal antibodies, which are homogeneouspopulations of antibodies to a particular antigen, may be obtained byany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited tothe hybridoma technique of Kohler and Milstein, (e.g. U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (e.g. Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (e.g. Cole et al., 1985, Monoclonal Antibodies And CancerTherapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (e.g. Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable orhypervariable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (e.g. U.S. Pat. No. 4,946,778) can be adapted to producedifferentially expressed gene-single chain antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide. Most preferably, techniques useful for the production of“humanized antibodies” can be adapted to produce antibodies to thepolypeptides, fragments, derivatives, and functional equivalentsdisclosed herein. Such techniques are disclosed e.g. in U.S. Pat. No.5,770,429, the disclosures of which are incorporated by reference hereinin their entirety. Antibody fragments which recognize specific epitopesmay be generated by known techniques.

Preferred antibodies of the present invention are expected to have,inter alia, similar biological functions/properties to a previouslydisclosed antibody (=IN-1) for the ligand of NgRH1)(see e.g. Schnell andSchwab, 1990, Nature 343, 269-272). Furthermore, preferred polypeptidesand polynucleotides of the present invention have at least one activityof IN-1. An array of oligonucleotides probes comprising the GBRSpolynucleotide sequence or fragments thereof can be constructed toconduct efficient screening of e.g., genetic mutations. Such arrays arepreferably high density arrays or grids. Array technology methods arewell known and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability, see, for example, M. Chee etal., Science, 274, 610-613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide ormRNA expression may also be used for diagnosing or determiningsusceptibility of a subject to a disease of the invention. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, nucleic acid amplification, for instance PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those skilled in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit comprising:

-   (a) a polynucleotide of the present invention, preferably the    nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 24, or a fragment    or an RNA transcript thereof;-   (b) a nucleotide sequence complementary to that of (a);-   (c) a polypeptide of the present invention, preferably the    polypeptide of SEQ ID NO: 2 or SEQ ID NO: 25 or a fragment thereof;    or-   (d) an antibody to a polypeptide of the present invention,    preferably to the polypeptide of SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlydiseases of the invention, amongst others.

A further embodiment of the invention relates to methods to identifycompounds that stimulate or inhibit the function or level of thepolypeptide. Accordingly, in a further aspect, the present inventionprovides for a method of screening compounds to identify those thatstimulate or inhibit the function or level of the polypeptide (e.g.blocking or stimulating NIH 3T3 cell spreading in vitro, blocking andstimulating PC12 neurite growth, inducing or blocking dorsal rootganglia growth cone collapse, spreading or blocking of neural cells,regeneration of lesioned nerve fibers in in vivo models). Such methodsidentify agonists or antagonists that may be employed for therapeuticand prophylactic purposes for such diseases of the invention ashereinbefore mentioned. Compounds may be identified from a variety ofsources, for example, cells, cell-free preparations, chemical libraries,collections of chemical compounds, and natural product mixtures. Suchagonists or antagonists so-identified may be natural or modifiedsubstrates, new ligands etc., as the case may be, of the polypeptide; astructural or functional mimetic thereof (see Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991)) or a small molecule.

The method may simply be a method of identifying a compound thatmodulates NgRH1 receptor activity, comprising:

-   (a) combining the compound with a NgRH1 receptor, preferentially    human NgRH1, most preferentially a receptor comprising the amino    acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO: 25; and-   (b) measuring an effect of the compound on the receptor.

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof, by means of a label directlyor indirectly associated with the candidate compound. Alternatively, thescreening method may involve measuring or detecting (qualitatively orquantitatively) the competitive binding of a candidate compound to thepolypeptide against a labelled competitor (e.g. agonist or antagonist).Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring a NgRH1 binding or activity inthe mixture, and comparing the NgRH1 binding or activity of the mixtureto a control mixture which contains no candidate compound. Polypeptidesof the present invention may be employed in conventional low capacityscreening methods and also in high-throughput screening (HTS) formats.Such HTS formats include not only the well-established use of 96- and,more recently, 384-well micotiter plates but also emerging methods suchas the nanowell method described by Schullek et al, Anal Biochem., 246,20-29, (1997).

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents that may inhibit or enhance theproduction of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of antibodies to NgRH1s,mimetics, agonists, antagonists, or inhibitors of NgRH1s. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions encompassed by the invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-articular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans. A therapeuticallyeffective dose refers to that amount of active ingredient, for exampleantibodies, agonists, antagonists or inhibitors of NgRH1, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED50 (the dose therapeutically effectivein 50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors that may be taken intoaccount include the severity of the disease state, general health of thesubject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc. Pharmaceutical formulations suitable fororal administration of proteins are described, e.g., in U.S. Pat. Nos.5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633;5,792,451; 5,853,748; 5,972,387; 5,976,569; and 6,051,561.

The following Examples illustrate the present invention, without in anyway limiting the scope thereof.

EXAMPLES Example 1 Isolation of the Human NgRH1 cDNA

Identification of human NgRH1 and sequence analysis—Using the publishedNgR amino add sequence (accession number: AF283463) as a query, tblastnsearch brought up two sequences from the Celera database, hCT31020 andGA_(—)53256868, the translational products of which showed roughly 50percent similarity (blast result) to the published NgR sequence.Alignments were performed, using either GAP or BESTFIT analysis from theWisconsin Package Version 10.1, Genetics Computer Group (GCG), Madison,Wis. Multiple sequence analysis were done by ClustalW. Signal peptideprediction for NgR and the homologues were done using Spscan.Leucine-rich-repeats (LRR) were identified through comparing sequenceswith various consensus segments present in the protein-family-databasePfam. Further identification and confirmation of LRR's were done byBLOCKS+alignments using the IMPALA software. Prediction of a potentialGPI cleavage site was done using DGPI, from the ExPaSy program packagepresent on the server from the Swiss institute of Bioinformatics (SIB).

Isolation of full length cDNAs encoding human NgRH1: The cDNA for theNgR homologue-1 was obtained by PCR from a human brain cDNA(Marathon-Ready™ cDNA, CLONTECH Laboratories, Inc., Palo Alto, Calif.,cat. Nr. 7400-1). The 5′- and the 3′ end of the gene were amplifiedseparately (see below). Sequence specific primers were designed based ona continuous sequence assembled from a predicted transcript sequencefrom the Celera database (accession number: hCT31020) and two ESTsequences from public DNA data bases (accession numbers: A1929019 andBE410139). PCRs were carried out on a PerkinElmer GeneAmp 9600 cycler,using 4% DMSO and Herculase™ Enhanced DNA-Polymerase (Stratagene Europe,Amsterdam, Netherlands). The 5′end (798 bp) was amplified from totalbrain Marathon-Ready™ cDNA (Clontech Laboratories, Inc., Palo Alto,Calif., cat. Nr. 7400-1) by 5′-RACE. A first amplification was carriedout using the AP1 primer from the kit and a sequence specific primerGSP1 (5′-GTG GTT GGA GGA GGC CTG GAA GT-3′ (SEQ ID NO: 3)). Nested PCRusing the AP2 primer from the kit and sequence specific primer nGSP1(5′-GGC CAG GCT GTT GTT GAA CAG G-3′ (SEQ ID NO: 4)) resulted in a 798bp fragment which was ligated into the pCR-Blunt II-TOPO cloning vector(Invitrogen, Basel, Switzerland). The correct sequence was confirmed byautomated sequence analysis. The 3′end of the gene (804 bp) wasamplified from a human fetal brain cDNA (Marathon Ready, ClontechLaboratories, Inc., Palo Alto, Calif., cat. Nr. 7402-1) by PCR usinggene specific primers GSP2 (5′-GGT CAG CCT GCA GTA CCT CTA CC-3′ (SEQ IDNO: 5)) and NgRH1-3′ (5′-AGT CAG AGG TGG TGG GGC ACC AG-3′ (SEQ ID NO:6)). The PCR fragment was ligated into pCR TOPO TA-vector (Invitrogen,Basel, Switzerland) and sequenced. The full length cDNA was thenassembled by overlapping PCR using the primers NgRH1-5′ (5′-AGA TGC TGCCCG GGC TCA GGC G-3′ (SEQ ID NO: 7)) and NgRH1-3′ (see above). The PCRproduct was ligated into pCR-Blunt II-TOPO cloning vector. This finalplasmid is called hNgRH1-fl hereafter. Sequence analysis confirmed thepredicted sequence from the Celera database except a point mutation(silent) at position 642 (G→A) and an additional 51 bases inserted atposition 926 of the predicted sequence. The 51 bp insert was confirmedby the presence of another EST in Genbank (accession number: BE222737).Recently a sequence has been deposited in the GenBank database(accession number: AX411529) that matches to our NgRH1 sequence. Thehuman NgRH1 is 45% similar on the amino acid level to human NgR.

Not only the complete DNA-sequence for human NgRH1 (SEQ ID NO: 1) can beobtained with this approach (1263 bp coding for 420 amino acids with amolecular weight of 42 kDa), but also the disclosed other variants andfragments of the invention. As an alternative, the first step inobtaining NgRH1s may start with using the amino acid sequence of humanNgRH1.

Leucine-rich-repeats (LRR) are identified through comparing sequenceswith various consensus segments present in the protein-family-databasePfam. Further identification and confirmation of LRR's are done byBLOCKS+ alignments using the IMPALA software. Prediction of a potentialGPI cleavage site is done using DGPI, from the ExPaSy program packagepresent on the server from the Swiss institute of Bioinformatics (SIB).

Thus, Human NgRH1 appears to belong to the same family ofleucine-rich/proteoglycan proteins as NgR. Like human NgR, human NgRH1codes for also 8 LRR's flanked by a leucine-rich-repeat-N-terminus and aleucine-rich-repeat-C-terminus. In addition to the presence of a signalsequence at the N-terminus, human NgRH1 contains a short hydrophobicamino acid stretch at its C-terminus, typical for GPI-linked proteins(see also Example 3).

The rat gene can be obtained by an analogous method. The cDNA coding forrat-NgRH1 is amplified by PCR from a rat brain cDNA-library(Marathon-Ready cDNA, BD Clontech, Palo Alto). PCR is performedaccording to standard protocols using Herculase Enhanced DNA Polymerase(Stratagene Europe, Amsterdam, Netherlands). Primers are chosen based onthe sequence of the 5′-UTR of human NgRH1 (SEQ ID NO:1)(5′-TGAATCTGGACCCCGGGAGG-3′ (SEQ ID NO: 8)) and the rat EST-sequence,acc. # BE097332 (5′-TCCTCAGCGGAGAGATACCACCA-3′ (SEQ ID NO: 9)). Thefull-length cDNA was cloned into pCR-TOPO-Blunt (Invitrogen). Thefull-length cDNA for rat-NgRH1 with the human sequence is 87% identicalon DNA level and 88% on amino acid level.

Example 2 Human NgR and NgRH1 Expression and their BiochemicalCharacterization

Materials—Media: MEM-alpha-plus medium and OptiMEM I with Glutamax(Invitrogen, Basel, Switzerland). Primary antibodies: monoclonal anti-V5antibody (Invitrogen, Basel, Switzerland); monoclonal anti-HA antibody,clone HA-7 (SIGMA, Buchs, Switzerland). Secondary antibodies: PODconjugated anti-mouse or anti-rabbit IgG antibody (SIGMA, Buchs,Switzerland); Anti-mouse IgG, FITC conjugated antibody (SIGMA, Buchs,Switzerland). Complete™ (Roche Applied Science (Rotkreuz, Switzerland).

Methods:

a) Northernblots: In order to determine the tissue distribution of hNgRand hNgRH1 expression, a Multiple Tissue Northernblot (MTN, CLONTECHLaboratories, Inc., Palo Alto, Calif.) and a Multiple Tissue ExpressionArray (MTE array, CLONTECH Laboratories, Inc., Palo Alto, Calif.) washybridized with α-³²P-dCTP and α-³²P-dATP-radiolabeled human NgR andNgRH1 probe. The probes for the respective cDNAs were generated asfollows. NgR probe was generated by excision of pcDNA-Sport6-NgR byEcoRI/Xho-I cleavage. This clone was obtained from a human dorsal rootganglion (DRG) cDNA library (Life Technologies Inc., Rockville, Md.). Itwas identified through a blast search against library clone sequences,using the human NgR cDNA as a query. The cDNA insert of pcDNA-Sport6-NgRis 24 and 292 bp longer on the 5′end and 3′end respectively, compared tothe published sequence for NgR (accession number: AF283463). The NgRH1probe was generated by excision of hNgRH1-fl by EcoRI cleavage. Theresulting 1.8 kb and 1.3 kb cDNA inserts for NgR and NgRH1 respectivelywere gel purified (QIAEX II Gel Extraction Kit, QIAGEN AG, Basel,Switzerland) and 100 ng each was radiolabeled using High Prime DNALabelling Kit (Roche Biochemicals, Rothkreuz, Switzerland) in threeseparate labelling reactions. Unincorporated nucleotides were removed,using the NucTrap Probe Purification Columns (Stratagene Europe,Amsterdam, Netherlands). MTN or MTE membranes were hybridised witheither NgR or NgRH1 probe in ExpressHyb solution (CLONTECH Laboratories,Inc., Palo Alto, Calif.) according to the manufacturer instructions(except that the Cot-1 DNA was left out). A single band at 2.4 kb forhuman NgR and human NgRH1 Is detected. Highest mRNA expression of humanNgR and human NgRH1 is in the human brain. However, abundant expressionof human HgRH1 can be seen in human liver too. Both genes are expressedat low levels in other peripheral tissues, such as skeletal muscle,spleen, kidney, lung and placenta. In order to get a more detailed viewof potential spatial differences in brain expression, we also probedMTE's, carrying spotted RNAs from various brain regions. From thisanalysis, it appeared that both genes were differentially expressed indifferent brain areas. While they are strongly expressed in the cerebralcortex, amygdala, hippocampus and accumbens nucleus, only NgR is highlyabundant in the cerebellum, compared to expression in the cortex. Commonto both genes is their weak expression in pons, corpus callosum, caudatanucleus, medulla oblongata, putamen, substancia nigra and spinal cord.

b) Cell culture and transfection: CHO-K1 cells are grown inMEM-alpha-plus medium. This medium is supplemented with 10% Fetal CalfSerum (FCS) final concentration and Penicillin Streptomycin to a finalconcentration of 200 U/ml. For transient and stable cell transfection,FUGENE 6 (Roche Applied Science, Rotkreuz, Switzerland) is usedaccording to the manufacture instructions. Cells expressing human NgRand human NgRH1 (pSecTag2B vector) were put under selection with Zeocinto a 0.25 mg/ml final concentration.

c) Construction of epitope tagged NgR and NgRH1—1.) NgR-V5 tag cloningprocedure: Two complementary, synthetic oligonucleotides (Microsynth,Balgach, Switzerland) 5′-CCG GTA AGC CTA TCC CTA ACC CTC TCC TCG GTC TCGATT CTA CGT CTA GAT ATC CTC GAG-3′ (SEQ ID NO: 10) and 5′-GAG CTC CTATAG ATC TGC ATC TTA GCT CTG GCT CCT CTC CCA ATC CCT ATC CGA ATG GCCCGA-3′ (SEQ ID NO: 11), coding for the V5-tag and restriction cleavagesites XbaI/EcoRV/XhoI were annealed and ligated into the SfiI-PmeI sitesof pSecTag2B vector (INVITROGEN, Basel, Switzerland) to get pSecTag2-V5.After this, the cDNA sequence coding for human NgR, without the signalpeptide, was amplified by PCR from pcDNA-Sport6-NgR (see above) usingforward primer 5′-GCA GCA TCT AGA CCA GGT GCC TGC GTA TGC TAC AAT GAGCCC-3′ (SEQ ID NO: 12) and reverse primer 5′-GCA GCA CTC GAG TCA GCA GGGCCC AAG CAC TGT CCA CAG CAC-3′ (SEQ ID NO: 13), cleaved with XbaI andXhoI and ligated into the respective cleavage sites in pSecTag2-V5. 2.)NgRH1-HA tag cloning procedure: Two complementary, syntheticoligonucleotides (Microsynth, Balgach, Switzerland) 5′-CCG ATT ACA AGGATG ACG ACG ATA AGT CTA GAC AGT GCG ATA TCA ATG AAT TC-3′ (SEQ ID NO:14) and 5′-CTT AAG TAA CTA TAG CGT GAC AGA TCT GAA TAG CAG CAG TAG GAACAT TAG CCC GA-3′ (SEQ ID NO: 15), coding for the FLAG-tag andrestriction cleavage sites XbaI/EcoRV/EcoRI were annealed and ligatedinto the SfiI-PmeI sites of pSecTag2B vector (INVITROGEN, Basel,Switzerland) to get pSecTag2-FLAG. After that, the cDNA sequence codingfor human NgRH1, without the signal peptide, was amplified fromhNgRH1-fl by PCR using the forward primer 5′-AAT TGC GCA TCT AGA GCC CCCAGC TGC CCC ATG CTC TGC ACC TGC-3′ (SEQ ID NO: 16) and reverse primer5′-AAT TGC GCA GAA TTC TCA GAG GTG GTG GGG CAC CAG CAG CAG GAG-3′ (SEQID NO: 17), cleaved with XbaI and EcoRI and ligated into the respectivecleavage sites in pSecTag2-FLAG.

Following stable cell transfection into CHO-K1 cells, human NgR andNgRH1 tagged with V5- or HA-tags respectively, were analysed inWesternblots. The respective cell lines expressed proteins larger thanthe molecular weights predicted for NgR (47 kDa) and NgRH1 (42 kDa), atarround 64 kDa. As we show later, the aberrant molecular weights can beexplained by post-translational modification. At least two major formsfor NgR are produced by the CHO cells. A protein band seen at 64 kDa,most likely corresponds to the full length NgR. The other one, atapproximately 48 kDa, seems to be a truncated NgR molecule. The 48 kDaband however wasn't detectable with the polyclonal α-NgR antisera. Thisis either due to the unspecific band running close to it, that makes theidentification impossible, or this form of NgR lacks epitopes againstwhich the antisera was raised. In contrast, the individual protein bandsfor NgRH1 could be confirmed with the specific polyclonal antisera. Withthe exception of α-NgR, none of the anti-sera picked up proteins in theuntransfected control cells, nor did they cross-react to different NgRspecies.

d) Cell surface expression of NgR and NgRH1—1.) Antibodies andimmunological detections—For the detection of NgR and NgRH1 proteins,either commercially available monoclonal anti-tag antibodies orpolyclonal antisera, raised in rabbits, were used. Generation ofpolyclonal antisera: Rabbit anti-NgR antisera was obtained form M.Schwab, University of Zürich raised against three synthetic peptidesfrom human NgR (EQLDLSDNAQLRSVDPA (SEQ ID NO: 18), EVPCSLPQRLAGRDLKR(SEQ ID NO: 19) and GPRRRPGCSRKNRTRS (SEQ ID NO: 20)) and affinitypurified by Research Genetics (Invitrogen, Corporation). Rabbit NgRH1antisera are raised against synthetic peptides (CPPMPTRPGSRARGN (SEQ IDNO: 21), DLPAEDSRGRQGGDAP (SEQ ID NO: 22) and TEDDYWGGYGGEDQR (SEQ IDNO: 23)) derived from the human NgRH1 sequence and affinity purified byEUROGENTECS (Seraing, Belgium). 2.) Westernblots: SDS-gelelectrophoresis were done using NuPAGE precast 4-12% Bis-Tris gels(INVITROGEN, Basel, Switzerland). Usually, MOPS running was used for theseparation. Electroblotting of the gels was done using a SemiphorTransphor Unit (Amersham Biosciences Dübendorff, Switzerland), applying24 V for 1 h. For immunodetection, the PVDF membranes were blocked for45 min in 5% skimmed milk in TBST, followed by a one hour incubationwith the primary antibody and secondary anti-mouse or anti-rabbit IgGantibody respectively, diluted in blocking solution. Membranes werewashed three times after each antibody incubation in TBST, containing 10mM Tris pH 7.5, 140 mM NaCl and 0.2% Tween 20, followed by a single washin TBS. Signals were developed using ECL™ Western Blotting DetectionReagents (Amersham Biosciences, Dübendorf, Switzerland) and Hyperfilm™ECL™ (Amersham Biosciences, Dübendorf, Switzerland) according to themanufacturer's instructions. 3.) Cell surface biotinylation: Cells atconfluency were incubated for 30 minutes at 4° C. with PBS containing 1mg/ml biotin 3-sulfo-N-hydroxysuccinimide ester (Sulfo-NHS-Biotin),rinsed three times with PBS containing 50 mM glycine and lysed in M-PER.Clarified lysates were incubated for 2 hr at room temperature with 20μg/ml streptavidin coupled to agarose beads and the beads subsequentlywashed successively with 10 mM Tris-HCl pH 7.8, 1% (w/v)N-lauroylsarcosine, 100 mM NaCl prior to SDS-PAGE analysis. NgR andNgRH1 are readely biotinylated on the cell surface of stable CHO-K1transfectants, whereas the control protein (GAPDH) is not. 4.)Immunoflourescence: For surface versus cytoplasmic staining, cells wereeither directly incubated with the primary antibody and then post-fixed,or cells were fixed in 4% paraformaldehyde and blocked in 10% FCS, 0.1%Triton X-100 for 20 minutes, prior to the primary antibody was added. AFITC coupled anti-mouse IgG antibody was used as secondary antibody.Pre-immune sera were used as controls and did not reveal unspecificbackground staining of intact cells.

In order to characterize the subcellular distribution of NgR and NgRH1and to show that all three proteins are cell surface expressed, weperformed cell surface biotinylation. Whereas no labeling of cytoplasmiccontrol protein GAPDH was seen, NgR and NgRH1 molecules were readilybiotinylated with the non-penetrable reagent Sulfo-NHS-Biotin, added tothe cells. The cell surface expression of NgR and NgRH1 was confirmed byimmunoflourescence of non-permeabelized, stable CHO cells, using eitheranti-tag antibodies or polyclonal antisera against the individual NgRproteins.

e) GPI-linkage and glycosylation of NgR and NgRH1: In the next couple ofexperiments we checked for post-translational modifications in vitro.Since a GPI-linkage of NgRH1 is predicted from their primary structures,we first checked this experimentally using PI-PLC. First, total cellextracts were incubated in the presence or absence of bacterialPhosphatidyl-Inositol-Phospholipase-C (PI-PLC). The removal of aGPI-linker alters the mobility of proteins in the SDS-PAGE (Cardoso deAlmeida et al., (1983) Nature 302, 349-52; Stahl, N. et al, (1987) Cell51, 229-40; Littlewood, G. M. et al., (1989) Biochem. J. 257, 361-7).Proteins with a GPI-linker usually get a higher loading with SDSmolecules due to the hydrophobic interaction with the lipid chains ofthe GPI-linker. The net charge therefore becomes slightly more negativecompared to proteins from which the GPI-linker has been enzymaticallyremoved. Thus GPI-removal results in a characteristic up-shift of therespective protein in the SDS-PAGE, compared to untreated controlproteins that are run in parallel lanes. To make the interpretationeasier, cells were pretreated with Tunicamycin to reduce the proteincomplexity caused by glycosylation. Following this protocol, NgR, aswell as NgRH1 indeed showed the characteristic up-shift in the SDS-PAGEafter PI-PLC treatment, demonstrating that they are GPI-linked. Thesecreted form of NgR remains completely unaffected from the PI-PLCtreatment. To further confirm these findings, cultured CHO cellsexpressing NgR or NgRH1 were incubated with PI-PLC. Subsequently, theproteins collected from the conditioned medium were analyzed byWesternblots, versus proteins remaining on the cells. NgR and NgRH1 werereleased into the conditioned medium after 3 h PI-PLC treatment,confirming that all of them are GPI-anchored. Interestingly, even in theabsence of PI-PLC, there is considerable, constitutive secretion of allthree different proteins. This is most apparent in case of the NgRexpressing cell line, from which the truncated form of NgR (48 kDa) thatwas observed before, is substantially released into the medium. Smallerproteins, but also potentially full length species of NgRH1 aredetectable in the control conditioned medium as well. To rule out thatthese observations are specific to CHO cells, we confirmed the existenceof two major forms for NgR in transiently transfected COS-7 cells. Thereare bands seen in the SDS-PAGE emerging in the cell pellets of NgR andNgRH1 CHO cells, whose identification cannot be determined easily. Asimple explanation would be that they stem from incompletelyglycosylated NgR molecules. All NgR proteins contain putativeN-glycosylation sites (Asn-X-Ser/Thr) in their amino acid sequence.Incubating the cells with Tunicamycin, markedly reduced the molecularweights of bands specific for NgR and NgRH1, demonstrating that all NgRproteins become highly glycosylated. The molecular weights now matchnicely the predicted sizes for NgR and NgRH1, reflecting the unmodifiedproteins. For NgR, the smaller secreted form is still detectable,showing that this molecule is produced independently of glycosylation.Interestingly, whereas Tunicamycin readily prevented the release of NgRinto the medium, it was less effective in case of NgRH1. Relatively highamounts of NgRH1 are still released into the medium after PI-PLCtreatment, indicative of a longer half-life of this protein, compared toNgR. The Tunicamycin experiment clearly showed that NgR and NgRH1 areglycosylated and therefore we suggest that additional bands that cannotbe assigned unambiguously to secreted or mature molecules in the cellpellet fraction, stem from immature precursors that have not undergonefull post-translational modification. Interestingly, these immatureforms only show up if the cells were PI-PLC treated. This might reflectincreased de novo protein synthesis after removal of NgR proteins fromthe plasma membrane, as compared to steady state conditions.

f) Lipid raft association of NgR and NgRH1—1.)Phosphatidylinositol-specific phopholipase C (PIPLC) treatment: i)Treatment of intact cells: Cells at confluency were washed twice inOptiMEM and then incubated for 4 hrs at 37° C. in 4 ml OptiMEMcontaining 0.2 U/ml PI-PLC (GLYKO Inc., Novato, Calif.). After 5 minutescentrifugation at 3000 rpm, the cell medium was concentrated six timesin a Centricon YM-10 (Millipore, Volketswil, Switzerland). The remainingcells were washed twice with PBS and harvested with a cell scraper andcentrifuged for 1 min at 5000 rpm. Cell lysis: Cell pellets were lysedin M-PER (100 μl per 25 mg cell pellet), sublemented with Complete™(ROCHE Applied Science, Rothkreuz, Switzerland) for 20 minutes at roomtemperature. After this, the lysed material was centrifuged for 10minutes at 14000 rpm; 4° C. and sample buffer was added to supernatant.Equivalent volumes of lysate and concentrated medium were subjected toSDS-PAGE (Invitrogen 4-12% Bistris Gels, MOPS buffer) and blotted ontoPVDF membranes. PI-PLC readily releases NgR and NgRH1 from transfectedCHO-K1 and 293T cells into the conditioned medium after PI-PLCtreatment, showing that they possess a GPI-anchor. ii) Treatment of celllysates: Cells were grown to 50%-80% confluency. The medium wasdiscarded and the cells were incubated at 37°; 5% CO₂ overnight in 3 mlOptiMEM (INVITROGEN, Basel, Switzerland) containing 5 μg Tunicamycin(GLYKO Inc., Novato, Calif.) per ml medium. The cells were then washedwith PBS and harvested by scraping. The harvested cells were centrifugedfor 1 min at 4° C. at 20000×g and lysed for 20 minutes at roomtemperature, in 200 μM-PER/Complete with EDTA per 100 mg of cell pellet.The sample was again centrifuged for 10 minutes at 4° C. with 20000×gand the supernatant was collected. This material was splited into twoequal aliquots and diluted 1:2 with 10 mM Hepes pH 7.6 buffer. To one ofthe aliquots 1 U/ml PI-PLC was added and both aliquots were incubate forat least 3 h at 37° C. Following PI-PLC treatment of cell lysates, NgRand NgRH1 show a characteristic upshift on SDS-PAGE indicative of theremoval of the GPI anchor (Cardoso et al. (1983) Nature 302, 349-52;Stahl et al. (1987) Cell 51, 229-40; Littlewood et al. (1989) Biochem.J. 257, 361-7). Lipid raft isolation: Lipid raft preparation was carriedout after Brown and Rose 1992 (Brown et al. (1983) Nature 302, 349-52).Briefly, cells at confluency from a 10 cm dish were washed with MBS (25mM MES-buffer/0.15M NaCl, pH 6.5) and scraped into same buffer. Aftercentrifugation at 1200 rpm for 5 minutes the cells were resuspended onice in 0.3 ml MBS. All following steps were carried out at 4° C. Thesample was adjusted to approx. 0.5 ml with MBS and to a finalconcentration of 1% Triton X-100 and homogenised in a Douncehomogeniser. Homogenised samples were mixed with equal volume of 80%(w/v) sucrose/MBS (0.5 ml) for final concentration of 40% (w/v) sucrose.The samples were then filled under the 5%/30% layers of sucrosegradients. Centrifugation of the gradients were carried out at 37 200rpm in SW50.1 rotor for 16-18 hr at 4° C. Lipid rafts/DRMs appear at the5%/30% interface as an opaque band. 9×0.5 ml fractions were harvestedfrom bottom to top of the gradient using a 1 ml syringe/21 G needle.

GPI-linked proteins are a characteristic constituent of lipid rafts.These microdomains of the plasma membrane are functionally andbiophysically distinct from the regular organization of the plasmamembrane structure, known as the phospolipid biolayer. Asymmetricalpacking of specific lipids, such as cholesterol and sphingolipids,determines a liquid-ordered state of rafts that result in aninsolubility in Triton X-100, at low temperature. This behavior has ledto the name detergent-resistant-membranes (DRM), which is used as atechnical term, synonymous to raft or caveolae. DRM's float to a lowdensity in sucrose gradients, due to their high lipid content. Thisenables any associated protein to be identified and distinguished fromother soluble components of the cells. Therefore, we extracted CHO cellsexpressing NgR and NgRH1 in 1% Triton X-100 at 4° C. and the resultingextracts were applied to sucrose density centrifugation. Under ourexperimental set up, sedimentation of DRM's usually takes place at the5%/30% sucrose interface (fraction 5 and 6) together with proteins knownto be associated with rafts, such as Flotillin. NgR and NgRH1 proteinsindeed sedimented in the expected fractions 5 and 6, together with themarker protein, demonstrating their lipid raft association. In contrast,the secreted form of NgR does not co-sediment with rafts, but smearsthrough several fractions from bottom to top of the sucrose gradient,mainly residing in high density fractions at 40% sucrose, containingTriton X-100 soluble material. This is also true for the secreted formsfrom NgRH1 that are found mainly in fractions 1 and 2. Two protein bandsfor Flotillin are detectable in the sucrose gradient, one band at 48 kDaand a smaller one at approximately 45 kDa. Both were originallydescribed by Bickel et al. (1997) J. Biol. Chem. 272, 13793-802). Thelower band at 45 kDa was found to be cross-reacting with the antibodyand is found in our hands in Triton X-100 soluble fractions of thesucrose gradient only.

Thus, we describe the characterization of proteins that were identifiedas homologues of the recently described receptor of Nogo-66. NgRH1 arehighly related to NgR in terms of primary structure, biochemicalproperties and expression pattern. Multiple lines of evidences aspresented above support the conclusion that NgR and the newly identifiedhomologue NgRH1 are members of a novel protein family.

Example 3 Ligand Binding Analysis

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand (putatively NogoA, NogoB, NogoC, Nogo-66, MAG of OMgp) for thereceptor hNgRH1 may be radiolabeled to high specific activity (50-2000Ci/mmol) for binding studies (or using suitable detection tags to theligands (agonists or antagonists) such as, alkaline phosphatase, GST,Myc, His, V5 etc). A determination may be then made that the process ofradiolabeling (or other signals) does not diminish the activity of theligand towards its receptor. Assay conditions for buffers, ions, pH andother modulators such as nucleotides may be optimized to establish aworkable signal to noise ratio for both membrane and whole cell receptorsources. For these assays, specific receptor binding may be defined astotal associated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand or in the presenceof an excess of the soluble NgRH1 ectodomain, lacking the GPI-anchor(Domeniconi et al. (2002) Neuron 35, 283-290 (published online June 28),Liu et al. (2002) Science June 27 (epub ahead of print). Where possible,more than one competing ligand may be used to define residualnonspecific binding.

Example 4 Functional Assays

Human NgRH1 may be expressed in recombinant expression systems such asHEK293 cells, CHO cells or COS cells and verified for expression at thecell surface (e.g. see Example 2d). Alternatively, hNgRH1 is expressedin recombinant expression systems as above together with putativeinteracting proteins (e.g. Nogo-66 or NogoA, NogoB, NogoC, MAG or OMgp).Co-transfection of cDNA expression constructs is for example done withthe Effectene transfection agent (Qiagen). A functional read-out mayinvolve analysis of agonist (e.g. by application or co-expression ofNogo-A,B,C, MAG or OMgp to CHO cells stably expressing NgRH1) inducedchange in cell adhesion, cell sprouting, intracellular CAMP levels andintracellular Ca²⁺ levels.

Alternatively, effect of compounds, antibodies and molecules blocking ordown-regulating the receptors is assessed and confirmed in standardfunctional assays for growth cone collapse, neurite outgrowth andspreading of 3T3 cells in the presence of Nogo ligands (e.g. Nogo A orC) as described in the following papers (Chen et al., 2001, Nature 403,434-439; Fournier et al., 2001, Nature 409, 341-346). Regenerativeeffects of these therapeutic agents is also be assessed in in vivomodels of brain and spinal injury as described e.g. in the followingpaper (e.g. Schnell et al., 1990, Nature 343, 269-272) and effect onfunctional deficits (e.g. Thallmair et al., 1998, Nature Neurosci. 1,124-131; Z'Graggen et al., 1998, J. Neurosci. 18, 4744-4754).

1. An isolated polypeptide selected from one of the groups consistingof: (a) an isolated polypeptide encoded by a polynucleotide comprisingthe sequence of SEQ ID NO: 1 or SEQ ID NO: 24; (b) an isolatedpolypeptide comprising a polypeptide sequence having at least 95%identity to the polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 25;(c) the polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 25 and (d)fragments of such polypeptides in (a) to (c).
 2. The isolatedpolypeptide according to claim 1, wherein the polypeptide sequence isSEQ ID NO: 2 or SEQ ID NO:
 25. 3. An isolated polynucleotide selectedfrom one of the groups consisting of: (a) an isolated polynucleotidecomprising a polynucleotide sequence having at least 95% identity to thepolynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 24; (b) anisolated polynucleotide comprising a polynucleotide sequence encoding apolypeptide sequence having at least 95% identity to the polypeptidesequence of SEQ ID NO: 2 or SEQ ID NO: 25; (c) an isolatedpolynucleotide comprising a nucleotide sequence of at least 100nucleotides; (d) a polynucleotide which is the RNA equivalent of apolynucleotide of (a) to (d); or a polynucleotide sequence complementaryto said isolated polynucleotide and polynucleotides that are variantsand fragments of the above mentioned polynucleotides or that arecomplementary to above mentioned polynucleotides, over the entire lengththereof. (e) an isolated polynucleotide which hybridizes under stringenthybridization conditions to SEQ ID No. 1 or SEQ ID No. 24, complementsor fragments thereof;
 4. The isolated polynucleotide according to claim3 wherein said polynucleotide sequence is 30 nucleotides, 30-50nucleotides, 50-100 nucleotides or 100-200 nucleotides in length.
 5. Anexpression vector comprising a polynucleotide capable of producing apolypeptide of claim 1 when said expression vector is present in acompatible host cell.
 6. A recombinant host cell comprising theexpression vector of claim
 5. 7. A process for producing a polypeptideof claim 1 comprising the step of culturing a host cell under conditionssufficient for the production of said polypeptide and recovering thepolypeptide from the culture medium.
 8. A fusion protein comprising theImmunoglobulin Fc-region and a polypeptide of claim
 1. 9. An antibodyimmunospecific for the polypeptide of claim
 1. 10. A method foridentifying a compound that modulates NgRH1 receptor activity,comprising: (a) combining the compound with a polypeptide according toclaim 1; and (b) measuring an effect of the compound on the receptor.11. The method according to claim 10, wherein said NgRH1 receptor is ahuman NgRH1 receptor comprising the amino acid sequence as set forth inSEQ ID NO:
 2. 12. A purified antibody or a fragment thereof whichspecifically binds to a polypeptide that comprises the amino acidsequence set forth in SEQ ID NO: 2 or SEQ ID NO: 25 or a fragment of apolypeptide that comprises the amino acid sequence set forth in SEQ IDNO: 2 or SEQ ID NO:
 25. 13. An antibody fragment according to claim 12which is an Fab or F(ab′)₂ fragment.
 14. The antibody according to claim12, which is a polyclonal antibody.
 15. The antibody according to claim12, which is a monoclonal antibody.